Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts (original) (raw)
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Letters in Applied Microbiology, 2002
Aims: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei. Methods and Results: Humicola grisea xylanase 2 (xyn2) cDNA was expressed in Trichoderma reesei under the main cellobiohydrolase I (cbh1) promoter (i) as a fusion to the cellobiohydrolase I (CBHI) secretion signal and (ii) the mature CBHI core-linker. The recombinant xylanase (HXYN2) was secreted into the cultivation medium and processed in a similar fashion to the endogenous T. reesei xylanases, resulting in an active enzyme. Conclusions, Signi®cance and Impact of the Study: HXYN2 was successfully processed in T. reesei. Composition of the culture medium affected the HXYN2 yields, favouring Avicellactose as a carbon source. Best yields (about 0á5 g l )1 ) in shake¯ask cultivations were obtained from a transformant where xyn2 was fused directly to the CBHI secretion signal.
Journal of Fungi
Trichoderma sp. is extensively applied as a beneficial fungus for the management of plant diseases, plant growth promotion, induced resistance, and plays an important role in global sustainable agriculture. This study aimed to enhance the production of microbial xylanase in high titer from the endophytic fungus Trichoderma harzianum kj831197.1, and the cloning of xylanase genes in E. coli DH5α using a pUC19 vector. A combination of glucose, 0.1 mM, Tween 80 with lactose, and 2 mM galactose combined with malt extract boostedthe enzyme production. Xylanase production was maximized at a pH of 5.0, temp. of 30 °C, and agitation of 150 rpm in the presence of malt extract and bagasse as the best nitrogen source and waste, respectively, using submerged fermentation. The molecular weight of highly purified xylanase was 32 KDa, identified using SDS-PAGE. The xylanase gene of T. harzianum kj831197.1 was screened in fungal DNA using definite primers specified in the gene bank database. The ide...
Applied and Environmental Microbiology, 2007
We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3 to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBDtruncated N. flexuosa xyn11A produced about 1.9 g liter ؊1 of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.
Partial Purification and Characterization of a Xylanase from Trichoderma Harzianum
Journal- Chemical Society of Pakistan
Among the fungi, the soft rot fungus Trichoderma has shown to be efficient producers of xylan degrading enzyme. In this paper we report partial purification and biochemical characterization of a xylanase (XYN) from a local isolate of a filamentous fungus Trichoderma harzianum. Xylanase was produced by growing the T. harzianum in Vogel’s medium where 1 % xylan was used as carbon source in medium to induce the production of enzyme. During purification 40% ammonium sulphate saturation was found to be optimal for xylanase precipitation. The crude sample was dialysed and was also followed by Sephadex G-200 chromatography steps. The specific activity of xylanase was increased about 3.14-fold compared with the crude preparations. The pH and temperature optima of the partially purified xylanase was 6.0 and 65 oC respectively. The apparent Km and Vmax values of the crude xylanase using birchwood xylan as a substrate were 1.52 mg mL-1 and 0.647 μmol min-1 mg-1, respectively.
FEMS Microbiology Letters, 2000
The catalytic domain of the xynB (xylanase) gene from the thermophilic bacterium Dictyoglomus thermophilum was reconstructed by PCR to match the codon preference of Trichoderma reesei. The 0.6-kb DNA fragment encoding the enzyme was first amplified by primer extension with a mixture of eight overlapping oligonucleotides, followed by PCR with outside primers containing restriction enzyme sites for directional cloning into Escherichia coli and T. reesei vectors. The synthetic gene was expressed in both organisms, producing a clearing halo around transformant colonies in plate assay utilizing an overlay of oat spelts xylan. Effective transcription of xynB in T. reesei was obtained after changing 20 codons. ß
Xylanase production by Trichoderma longibrachiatum
Enzyme and Microbial Technology, 1989
The effects of carbon source, substrate concentration, culture pH, and spore inoculum concentration on production of extracellular xylanase and cellulase were examined. Very low enzyme activities were obtained with growth on glucose, xylose, and cellobiose, while significantly higher levels were produced from lactose and arabinose. Higher levels of both enzymes were generated from alpha cellulose, wood pulp, and fibrous paper waste than from purified xylan. However, the ratio of xylanase to cellulase activity was higher with pure xylan. High levels of both xylanase and cellulase activity were obtained when the culture was grown on lactose plus xylan. A factorial experiment showed that spore and substrate concentration had significant effects on xylanase yield, and that an interaction existed between these two variables. Highest levels of xylanase were generated with cultures grown on 1% wood pulp at pH 7.0 using an inoculum of lO 5 spores ml -I. Maximal xylanase activity was observed at pH 4.8-5.5 at 55°C using a 30-min assay. The type of xylan used as substrate and the method of reducing sugar detection significantly affected measured xylanase activity.
Xylanase XYN IV from Trichoderma reesei showing exo- and endo-xylanase activity
FEBS Journal, 2013
A minor xylanase, named XYN IV, was purified from the cellulolytic system of the fungus Trichoderma reesei Rut C30. The enzyme was discovered on the basis of its ability to attack aldotetraohexenuronic acid (HexA-2Xyl-4Xyl-4Xyl, HexA 3 Xyl 3 ), releasing the reducing-end xylose residue. XYN IV exhibited catalytic properties incompatible with previously described endo-b-1,4-xylanases of this fungus, XYN I, XYN II and XYN III, and the xylan-hydrolyzing endo-b-1,4-glucanase EG I. XYN IV was able to degrade several different b-1,4-xylans, but was inactive on b-1,4-mannans and b-1,4-glucans. It showed both exo-and endo-xylanase activity. Rhodymenan, a linear soluble b-1,3-b-1,4-xylan, was as the best substrate. Linear xylooligosaccharides were attacked exclusively at the first glycosidic linkage from the reducing end. The gene xyn4, encoding XYN IV, was also isolated. It showed clear homology with xylanases classified in glycoside hydrolase family 30, which also includes glucanases and mannanases. The xyn4 gene was expressed slightly when grown on xylose and xylitol, clearly on arabinose, arabitol, sophorose, xylobiose, xylan and cellulose, but not on glucose or sorbitol, resembling induction of other xylanolytic enzymes from T. reesei. A recombinant enzyme prepared in a Pichia pastoris expression system exhibited identical catalytic properties to the enzyme isolated from the T. reesei culture medium. The physiological role of this unique enzyme remains unknown, but it may involve liberation of xylose from the reducing end of branched oligosaccharides that are resistant toward b-xylosidase and other types of endoxylanases. In terms of its catalytic properties, XYN IV differs from bacterial GH family 30 glucuronoxylanases that recognize 4-O-methyl-D-glucuronic acid (MeGlcA) substituents as substrate specificity determinants.
Purification and characterization of a new Xylanase from Humicola grisea var. Thermoidea
Brazilian Journal of Microbiology, 2004
The thermophilic fungus Humicola grisea var. thermoidea secretes extracellular xylanase when grown on solid and in liquid media containing wheat bran and banana plant residue as substrates, respectively. At 55ºC, xylanase from the culture filtrate of H. grisea var. thermoidea grown on banana stalk retained 50% of its activity after 28 h of incubation. A xylanase (X2) was isolated from solid state cultures with wheat bran as the carbon source. It was purified to apparent homogeneity by ultrafiltration followed by ion-exchange and hydrophobic interaction chromatography on DEAE-Sepharose and Phenyl-Sepharose resins, respectively. The enzyme had an apparent molecular weight of 29 kDa, as determined by SDS-PAGE. The purified enzyme was most active at pH and temperature ranges of 4.5-6.5 and 55-60ºC, respectively. In addition, X2 showed thermostability at 60ºC with a half-life of approx. 5.5 h. The apparent Km values, using soluble and insoluble arabinoxylans as substrates, were 10.87 and 11.20 mg/ml, respectively.